In the in vivo synthesis of proteins occurring in the ribosomes, mRNA is translated into polypeptide chains. However, the MRNA codons do not directly recognize the amino acids that they specify in the way that an enzyme recognizes a substrate. Translation uses "adaptor" molecules that recognize both an amino acid and a triplet group of nucleotide bases (a codon). These adaptors consist of a set of small RNA molecules known as transfer RNAs (or tRNAs), each of which is only 70 to 90 nucleotides in length. Such tRNA molecules contain unpaired nucleotide residues comprising a CCA triplet at one end of the molecule and, in a central loop, a triplet of varying sequence forming the so-called anticodon that can base-pair to a complementary triplet in the MRNA molecule, while the CCA triplet at the free 3' end of the molecule is attached covalently to a specific amino acid.
The three nucleotide triplets UAG (amber codon), UGA (opal codon) and UAA (ochre codon) do not code for an amino acid. These signals termed stop codons or "nonsense" codons, are involved in polypeptide chain termination. During translation, two protein factors (R1 and R2) recognize these triplets and effect release of the polypeptide chain from the ribosome-mRNA-tRNA complex.
Occasionally a mutation occurs in a cell resulting in a nonsense codon appearing in the middle of a gene, causing premature chain termination and the production of a protein fragment. Such fragments rarely have enzymatic activity.
The effect of such a nonsense mutation can be reversed or suppressed by a second mutation in a gene coding for a tRNA which results in the synthesis of an altered tRNA molecule. Such an altered tRNA recognizes a nonsense codon and inserts an amino acid at that point in the polypeptide chain. The mutated tRNA-encoding gene is termed a suppressor gene and the altered nonsense mutation-suppressing tRNA which it encodes is generally referred to as a nonsense or termination suppressor. Such termination suppressors may be derived by single, double or triple base substitutions in the anticodon region of the tRNA.
Termination suppressors were first detected in E. coli about 25 years ago and have since been extensively studied in this species. It is considered that all termination suppressors in E. coli have been identified. Recently, new suppressor tRNA genes have been synthesized in vitro and subsequently introduced into E. coli. Termination suppressors have also been identified in the E. coli bacteriophage T4 and in Salmonella typhimurium (Eggertson et al., 1988, Microbiological Reviews, 52, 354-374). Furthermore, termination suppressors have been identified in eucaryotic fungi including Neurospora spp., Saccharomyces cerevisiae and Schizosaccharomyces pombe.
Hitherto, nonsense or termination suppressors have not been identified in bacterial species belonging to the industrially important group of lactic acid bacteria which i.a. are commonly utilized as starter cultures in the production of a variety of food products including dairy products, meat products, vegetable products, bakery products and wine, during which production these starter cultures produce lactic acid and other organic acids and in many instances also desirable flavour-enhancing metabolites.
Furthermore, attempts by the present inventors to construct amber-suppressing strains of lactic acid bacteria by introducing cloned known suppressor genes from E. coli proved unsuccessful. Thus, it was attempted to introduce the E. coli supB gene (Thorbjarnadottir et al., 1985), the E. coli supE gene (Nakajima et al., 1981) and the E. coli supF gene (Ryan et al., 1980). These three suppressor genes were moved to pFDi3 described in the below Example 3 and analyzed for suppressor activity in Lactococcus lactis by testing for the expression of erythromycin resistance. None of the three E. coli suppressor genes expressed suppressor activity in Lactococcus lactis.
In many instances it is advantageous to use lactic acid bacterial starter cultures which are composed of two or more different species, since the metabolic activity of one species may enhance the growth of an other species or because different lactic acid bacterial species may have particularly advantageous effects on flavour development of the food product at specific stages of the food production.
Accordingly, an industrial need exists to provide mixed lactic acid bacterial starter cultures in which a particular characteristic is confined (or contained) to a particular strain. Commonly, genes coding for desired characteristics of a lactic acid bacterium are located on extrachromosomal replicons such as plasmids. It may therefore be advantageous to have such replicons contained in their original host species. As used herein, the term "contained" indicates confinement of a replicon to a specific host cell or to the stable maintenance of a replicon in a lactic acid bacterial host cell when this host cell is present in a specific environment. This stable maintenance in a particular host cell of a replicon may also be referred to as stabilization of that replicon.
The term "containment" may also as used herein as encompassing the phenomenon that the growth and/or viability of a specific lactic acid bacterial strain in a particular environment is controlled.
The known methods of stably maintaining (stabilizing) replicons to a host cell involve the insertion of relatively large DNA sequences such as a partitioning function. However, as it is well-known, the insertion of large DNA sequences involves the risk of deletion of other sequences from the replicon. It has now been found that nonsense suppressor-encoding lactic acid bacterial strains may be developed which provide the means of a novel and advantageous method of confining replicons to lactic acid bacterial strains. In contradistinction to the known methods of stabilizing (confining) replicons to host cells, the method as defined herein makes use of genes coding for suppressor TRNA which are small and may be inserted without causing deletions of desired genes.
In the production of food products where live microorganisms are used, it may be critical for the obtainment of the desired quality of the products that the microbial processes can be controlled effectively. This is particularly important when mixed starter cultures as defined above and which comprise a multiplicity of strains, are used. Such a control has hitherto been difficult to achieve since specific regulating mechanisms at the level of cell numbers and activity and at the level of gene expression in particular strains had to be selected individually for each of the strains used in the mixed culture. However, the present invention has made it possible that the same suppressor gene under the control of the same regulatory mechanism may be inserted in all of the strains of the mixed starter culture whereby the activity of the species of such a culture may be regulated concomitantly or, if different regulatory sequences are inserted in individual species members of the starter culture, the activity of the individual members may be regulated independently.